Myeloperoxidase from human leukocytes

Neutrophils obtained from the fluid leakage of the mouse peritoneal lavage were incubated with different doses of noreugenin (NRG: 1, 2.5, 5, 10, or 50 μM) or α-hydroxy-butein (AH-BU: 1, 2.5, 5, 10, or 50 μM) in the absence or presence of LPS (5 μg/mL) for 18 h in an EIE plate. The mixture was centrifuged at 900 × g for 5 min at 4°C (Sorvall™ ST 40, Thermo Scientific®, Swedesboro, NJ, USA). The neutrophil culture supernatant was used to measure MPO activity by a colorimetric assay, using the following procedure: the in-house assay method described in the literature was used to determine the MPO activity [24 (link)]. For this determination, 20 μL of each sample supernatant was added to 180 μL of buffer solution (composition: 0.167 mg/mL o-dianisidine dihydrochloride and 0.0005% H₂O₂), transferred to the EIE plate, and incubated at 37°C for 15 min. After this time, 15 μL of a stop solution (sodium azide, 1%) was added to the each well in the EIE plate. The colorimetric assay was performed using an EIE plate reader (Organon Teknika, Roseland, NJ, USA) at 450 nm and interpolated from a standard MPO curve (0.7–140 mU/mL, using myeloperoxidase from human leukocytes (Sigma-Aldrich, St. Louis, MO, USA)). The results were expressed in mU of MPO/mL, whereby 1 unit of MPO was defined as the amount of enzyme degrading 1 nmol H₂O₂ per min at 37°C.